Gene expression can be effectively regulated by modulating the rate of either transcription of DNA to messenger RNA (mRNA), or translation of the mRNA to the corresponding protein. Many genes include an inducible promoter region that specifically controls expression of the one or more genes operably linked to the promoter region. Transcription of these genes can be turned on or off in response to inducer molecules, including metabolic intermediates, changes in physical conditions including temperature changes, and exogenous molecules such as alcohol, or antibiotics. However, inducible promoters are not available for all physically relevant genes. Consequently, RNA-based gene control elements called riboswitches have attracted increasing attention and artificial riboswitches have been developed for a variety of purposes.
Riboswitches are mRNA-based regulatory devices that mediate ligand-dependent control of gene expression. A riboswitch includes an aptameric region that binds the inducer molecule (ligand), and causes a structural change in the expression platform portion of the mRNA riboswitch that in turn either increases or decreases expression of the corresponding gene. Hence, riboswitches regulate gene expression at the translational level. One example is a theophylline-responsive ON riboswitch for the csrA (carbon storage regulator) gene in Escherichia coli which provides flexible control of cellular auto-aggregation and motility of the resulting E. coli switch-csrA mutant organism. Riboswitches have also been used to regulate gene expression by mediating pre-mRNA splicing in the presence of an inducer molecule such as theophylline.
Ligand-dependent riboswitches are one-way switches, and, once activated, can be turned off only by removing the riboswitch ligand, rendering them impractical for in vivo applications. For in vitro applications, the growth medium must be diluted or changed to remove the riboswitch ligand. In addition, the riboswitches developed to date are specific to a given target gene and are not applicable to other genes. This lack of portability severely limits the applications suitable for these riboswitches.
It is desirable to obtain a portable riboswitch that provides two-way regulation in response to two distinct ligands. Such a riboswitch could be inserted upstream from the start codon of any target gene and exert two-way control of expression of the gene. Such a device would find a wide variety of applications including scientific studies into gene function, medical applications including gene therapy and applications in biotechnology.